Humans over the age of 65 contribute disproportionately to cold- and heat-related morbidities and mortalities and the relative risk of death from all causes increases during cold and hot weather. Because core temperature excursions during even mild heating or cooling (i.e., in the absence of shivering or sweating) are greater in the elderly, altered vasomotor regulation is the seminal age-related deficit. Recently we have examined the mechanisms of age-related attenuated reflex cutaneous vasoconstriction (VC) and vasodilation (VD). This attenuated reflex cutaneous vasoreactivity in aged skin is due to a functional loss of adrenergic VC and active cholinergic VD co-transmitter(s), and altered downstream vascular signaling including (1) adrenergic desensitization, and (2) decreased nitric oxide (NO) availability. The logical extension of that work, supported by provocative preliminary and pilot data, is to examine the role of oxidant-induced reduced tetrahydrobiopterin (BH4) bioavailability in the aged cutaneous vasculature. Human aging is associated with increased in oxidant stress leading to the destruction of the critical enzymatic cofactor BH4. BH4 is ubiquitous throughout neural and vascular tissues and has emerged as a viable molecular target that may underlie both attenuated reflex cutaneous VC and VD mechanisms. Age-related deficiencies in BH4 appear to play a central role in attenuated reflex cutaneous VC. In adrenergic nerve terminals, BH4 is required for the synthesis and packaging of catecholamines by serving as a critical cofactor for tyrosine hydroxylase and by maintaining the enzyme in its reduced active form. In Specific Aim 1 we will examine the role of reduced BH4 bioavailability in attenuated reflex VC in aged skin using physiological and pharmacological stimuli in separate experiments to induce sympathetic VC. BH4 is also an essential cofactor for the NO-synthase (NOS) isoforms which are necessary for the full expression of reflex cutaneous VD. NOS uncouples during substrate and/or oxidant-induced BH4 deficiencies resulting in superoxide production. Increasing substrate availability for NOS and/or providing supraphysiological doses of ascorbate augments NO-dependent VD in aged human skin. Because of the putative role of substrate availability in NOS uncoupling, and the redundancies in the mechanisms of action of ascorbate as (1) a non-specific antioxidant, (2) an inhibitor of S-nitrosylation of arginase, and (3) a chemical stabilizer/resynthesizer of BH4, we now aim to explore the role of BH4 deficiency in NOS uncoupling in aged human skin. In Specific Aim 2 we will determine the roles of BH4 and reduced substrate availability in the NOS uncoupling that results in attenuated NO-dependent reflex cutaneous VD in aged skin. In Specific Aim 3 we will test the efficacy of systemic oral BH4 supplementation strategies in improving reflex cutaneous VC and VD in aged human subjects during thermal stress (whole body cooling and heating; in separate experiments). PUBLIC HEALTH RELEVANCE: As a larger percent of the population surpasses retirement-age, the health issues of the elderly become pervasive exacting emotional, physical and financial costs. The results from this study will provide new and important information on the physiology governing impaired vasoconstriction and vasodilation in older humans that leads to thermal injury and death. Furthermore, results from these studies may provide an oral intervention strategy to functionally improve impaired cutaneous vasoreactivity in the aged during thermal stress.